This invention is in the field of molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding scorpion toxins that are sodium channel modifiers.
Alpha neurotoxins are short, single-chain, polypeptides responsible for insect and mammal poisonings. These neurotoxins show variability in their apparent toxicity, in their primary structures, and in their binding features to neuronal membrane preparations (Dufton and Rochat (1984) J. Mol. Evol. 20:120-127). Despite differences in their primary structures and phylogenetic selectivity, scorpion neurotoxins affecting sodium (Na) channels are closely related in their spatial arrangements and form a compact globular structure kept rigid by the four disulfide bridges (Miranda et al. (1970) Eur. J. Biochem. 16:514-523; and Fontecilla-Camps (1989) J. Mol. Evol. 29:63-67).
Zilbergberg and coworkers determined that single amino acid residues are important for receptor binding and for biological activity of scorpion Na-channel toxins (Zilbergberg et al. (1997) J. Biol. Chem. 272:14810-14816). As examples, the lysine at position 8 of LqhIT was demonstrated to be necessary for binding activity and toxicity without change in overall structure. A substantial decrease in biological activity without a significant change in structure was found when the aromatic amino acid phenylalanine, at position 17, was substituted for glycine. Conversely, changes in structure are not necessarily associated with differences in toxicity as demonstrated when tyrosine at position 49 was changed to leucine.
While potassium (K) channels have been shown to be central to heart function, the role of chlorine-(Cl) and Na-channels in this activity is less clear (Johnson et al. (1998) J. Neurogent. 12:1-24). Sodium entry hyperpolarizes the cell, producing indirect, Na-dependent changes of calcium transport (Friedman (1998) Annu. Rev. Physiol. 60:179-197). Abnormal influx of calcium is thought to be very important in the pathogenesis of several central nervous system disorders in vertebrates, including stroke damage, epilepsy, and the neuronal death associated with chronic epilepsy.
Excitatory amino acids, most notably glutamate and aspartate, are the predominant excitatory neurotransmitters in the vertebrate (including human) central nervous system. These amino acids are released from presynaptic nerve terminals and, after diffusing across the synaptic cleft, contact special receptor molecules in the postsynaptic cell membrane. These receptors indirectly influence the flow of various ions across the cell membrane and thus contribute to production of an electrical response to the chemical message delivered by neurotransmitter molecules. A number of common and very serious neurological problems involve the abnormal functioning of excitatory amino acid synapses. These include epilepsy, several degenerative disorders such as Huntington""s disease, and neuronal death following stroke. Unfortunately, there are very few chemical agents which are potent and selective blockers of excitatory amino acid receptors. Na-channel modifiers may be used for these purposes.
A drug with high affinity for the receptor could be expected to produce irreversible blockade of synaptic transmission. When labeled with some tracer molecule, such a drug would provide a reliable way of tagging receptors to permit measurement of their number and distribution within cells and tissues. These features would have very valuable consequences for research on excitatory amino acid neurotransmission and for the development of therapeutic agents to treat central nervous system dysfunction in humans and animals. Methods for treating heart and neurological diseases by applying toxins derived from spiders have been described (U.S. Pat. No. 4,925,664).
Arthropod animals, including insects, and certain parasitic worms use excitatory amino acids as a major chemical neurotransmitter at their neuromuscular junction and in their central nervous system. Because of the damage done by insect pests and the prevalence of parasitic worm infections in animals and humans in many countries, there is a constant need for potent and specific new pesticides and anthelmintic drugs that are non-toxic to humans, pets, and farm animals.
Chemical insecticides are an integral component of modem agriculture, and are an effective means for reducing crop damage by controlling insect pests. However, chemical agents are under continuous scrutiny due to the potential for environmental contamination, selection of resistant populations of agronomic pests, and toxicity to non-target organisms such as beneficial insects, aquatic organisms, animals and humans. As a result, alternative strategies for insect control are being sought that are effective and yet benign to non-target populations and the environment. One of these strategies is to use microorganisms that are naturally occurring pathogens of target insect populations. The expression of scorpion toxins using baculovirus vectors will be an advantage since these toxins have been previously shown to be highly toxic and very specific (Zlotkin et al. (1995) American Chemical Society, Symposium on Agrochemicals).
Due to a combination of problems associated with some synthetic insecticides, including toxicity, environmental hazards, and loss of efficacy due to resistance, there exists a continuing need for the development of novel means of invertebrate control, including the development of genetically engineered recombinant baculoviruses which express protein toxins capable of incapacitating the host more rapidly than the baculovirus infection per se.
Scorpion venoms have been identified as possible sources of compounds providing insecticidal properties. Two insect-selective toxins isolated from the venom of the scorpion Leiurus quinquestriatus and affecting sodium conductance have been reported previously (Zlotkin et al. (1985) Arch. Biochem. Biophys. 240:877-87). One toxin, AaIT, induced fast excitatory contractive paralysis of fly larvae and the other, LqhIT2, induced slow depressant flaccid paralysis suggesting that these two toxins have different chemical and pharmacological properties (Zlotkin et al. (1971) Biochimie (Paris), 53:1073-1078). Thus, other toxins derived from scorpion venom will also have different chemical and pharmacological properties.
The present invention concerns an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a first nucleotide sequence of at least 180 nucleotides selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 28, 30, 32, 34, and 36; (b) a second nucleotide sequence encoding a polypeptide of at least 60 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 29, 31, 33, 35, and 37; or (c) a third nucleotide sequence comprising the complement of the first or second nucleotide sequences.
In a second embodiment, this invention relates to an isolated polynucleotide encoding a mature scorpion Na-channel agonist.
In a third embodiment, this invention concerns an isolated polynucleotide comprising a nucleotide sequence of at least 60 (preferably at least 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 28, 30, 32, 34, and 36 and the complement of such nucleotide sequences.
In a fourth embodiment, this invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to at least one suitable regulatory sequence.
In a fifth embodiment, the present invention concerns a host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, an insect cell or mammalian cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.
In a sixth embodiment, the invention also relates to a process for producing a host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting a compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.
In a seventh embodiment, the invention concerns a scorpion Na-channel agonist polypeptide of at least 60 amino acids comprising at least 80% identity based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 29, 31, 33, 35, and 37.
In an eighth embodiment, the invention concerns a method of obtaining a nucleic acid fragment encoding a substantial portion of a scorpion Na-channel agonist polypeptide, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least 60 (preferably at least 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 28, 30, 32, 34, and 36, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a substantial portion of a scorpion Na-channel agonist amino acid sequence.
In a tenth embodiment, this invention relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a scorpion Na-channel agonist polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing a cDNA or genomic fragment that comprises the isolated DNA clone.
In an eleventh embodiment, this invention concerns a composition, such as a hybridization mixture, comprising an isolated polynucleotide or an isolated polypeptide of the present invention.
In a twelfth embodiment, this invention concerns a method for expressing a gene encoding Na-channel agonist in the genome of a recombinant baculovirus in an insect cell culture or in viable insects wherein said insect cells or insects have been genetically engineered to express an AAH IT4, an LqhIT2 precursor, an IT-2 precursor, a TsnTxp, an insecticidal toxin, a BmK M1 precursor, a neurotoxin V-5, or an AS neurotoxin. The recombinant baculovirus expression vector comprising a DNA sequence encoding a polypeptide of at least 60 amino acids comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 29, 31, 33, 35 and 37.